The formation and function of microtubule structures depends on dynamic instability, the asynchronous slow growth and rapid shrinkage of microtubules under equilibrium conditions. A complete understanding of dynamic instability will be valuable in determining how cells regulate microtubules, and should provide insights for the development of antimitotic drugs for chemotherapeutic uses. Dynamic instability arises from the intrinsic properties of the alpha/beta tubulin dimers. Beta- tubulin hydrolyzes GTP upon assembly into microtubules. This assembly- dependent GTP hydrolysis is essential for the rapid shrinkage of microtubules. It is not known how assembly stimulates GTP hydrolysis. This research proposal tests the hypothesis that alpha-tubulin stimulates GTP hydrolysis by interdimer contact with beta tubulin upon assembly. Yeast tubulin mutants, altered at dimer-dimer interfaces, will be examined for their effects on microtubule dynamics and GTP hydrolysis. Microtubule dynamics will be visualized in living cells by GFP-labeling and epifluorescence microscopy. Mutant tubulins that alter microtubu1e dynamics will be purified and their GTPase activity assayed during assembly. Among the mutants that will be tested, the alpha- tubulin mutant tub1-828 will be studied for the following reasons: (i) it contains a mutation of glutamate-255 (E255), which likely interacts with beta-tubulin-bound GTP only in assembled protofilaments, (ii) mutation of the E255-cognate residue in the tubulin-like bacterial protein FtsZ eliminates GTPase activity without affecting GTP binding. A beta-tubulin mutant with accelerated GTPase activity, tub2-T1O7K, will be tested to see if it suppresses tub1-828. The two mutations in tub1-828, E255A and D232A, will be separated and examined individually to define the function of each residue. The results of these studies will define the currently unknown function of alpha-tubulin in dynamic instability and establish a heretofore missing link between altered in vivo microtubule dynamics and the overall phenotypes of tubulin mutants in yeast.